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Dissecting epigenetic silencing complexity in the mouse lung cancer suppressor gene Cadm1.

Reamon-Buettner SM, Borlak J - PLoS ONE (2012)

Bottom Line: Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications.Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders.Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Environmental Hygiene, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany. reamon-buettner@item.fraunhofer.de

ABSTRACT
Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and 'bivalent' histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

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Chromatin analysis with micrococcal nuclease (MNase) to map nucleosome positions in Cadm1 promoter region.(A) Position of five predicted nucleosomes using the Segal algorithm, the location of PCR primers used in amplifying fragments after digestion of chromatin with MNase, and MNase-preferred sites (CATA). Fragments that were also analyzed by quantitative-PCR are boxed. (B) Quantity of amplified fragments in different lung cancer cell lines, including two cell lines that were treated with 5-aza-dC, and a ’blind‘ control uncharacterized cell line (AEII) which does not express Cadm1. BD10-aza in nuc2F3-1/2R3-1 is a missing value. (C) Mouse normal lung, mouse lung tumor as compared to lung cancer cell lines, A2B1 and A2C12. The chromatin here analyzed for A2B1 and A2C12 are different from those in (B).
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pone-0038531-g004: Chromatin analysis with micrococcal nuclease (MNase) to map nucleosome positions in Cadm1 promoter region.(A) Position of five predicted nucleosomes using the Segal algorithm, the location of PCR primers used in amplifying fragments after digestion of chromatin with MNase, and MNase-preferred sites (CATA). Fragments that were also analyzed by quantitative-PCR are boxed. (B) Quantity of amplified fragments in different lung cancer cell lines, including two cell lines that were treated with 5-aza-dC, and a ’blind‘ control uncharacterized cell line (AEII) which does not express Cadm1. BD10-aza in nuc2F3-1/2R3-1 is a missing value. (C) Mouse normal lung, mouse lung tumor as compared to lung cancer cell lines, A2B1 and A2C12. The chromatin here analyzed for A2B1 and A2C12 are different from those in (B).

Mentions: We were able to amplify fragments for the five predicted nucleosomes in MNase-digested chromatin in normal lung, lung tumor and in the different lung cancer cell lines, suggesting nucleosomal nature of DNA (Figure 4). Primers designed to amplify products inside nucleosomes exhibited higher efficiency (banding intensity and/or qPCR values), than those primers that amplify bigger products and/or shifted to the left or right borders of nucleosomes. Furthermore, amplification efficiency differed among the lung cancer cell lines and was generally higher in cell lines which were less methylated and still expressed Cadm1. Such differences became more evident with the left or right border primers that in some cell lines amplification products were already absent. This result suggested differential nucleosome positioning among the different lung cancer cell lines. Overall, the highest amplification efficiency was observed in normal lung, then in lung tumor, and followed by the lung cancer cell lines.


Dissecting epigenetic silencing complexity in the mouse lung cancer suppressor gene Cadm1.

Reamon-Buettner SM, Borlak J - PLoS ONE (2012)

Chromatin analysis with micrococcal nuclease (MNase) to map nucleosome positions in Cadm1 promoter region.(A) Position of five predicted nucleosomes using the Segal algorithm, the location of PCR primers used in amplifying fragments after digestion of chromatin with MNase, and MNase-preferred sites (CATA). Fragments that were also analyzed by quantitative-PCR are boxed. (B) Quantity of amplified fragments in different lung cancer cell lines, including two cell lines that were treated with 5-aza-dC, and a ’blind‘ control uncharacterized cell line (AEII) which does not express Cadm1. BD10-aza in nuc2F3-1/2R3-1 is a missing value. (C) Mouse normal lung, mouse lung tumor as compared to lung cancer cell lines, A2B1 and A2C12. The chromatin here analyzed for A2B1 and A2C12 are different from those in (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368868&req=5

pone-0038531-g004: Chromatin analysis with micrococcal nuclease (MNase) to map nucleosome positions in Cadm1 promoter region.(A) Position of five predicted nucleosomes using the Segal algorithm, the location of PCR primers used in amplifying fragments after digestion of chromatin with MNase, and MNase-preferred sites (CATA). Fragments that were also analyzed by quantitative-PCR are boxed. (B) Quantity of amplified fragments in different lung cancer cell lines, including two cell lines that were treated with 5-aza-dC, and a ’blind‘ control uncharacterized cell line (AEII) which does not express Cadm1. BD10-aza in nuc2F3-1/2R3-1 is a missing value. (C) Mouse normal lung, mouse lung tumor as compared to lung cancer cell lines, A2B1 and A2C12. The chromatin here analyzed for A2B1 and A2C12 are different from those in (B).
Mentions: We were able to amplify fragments for the five predicted nucleosomes in MNase-digested chromatin in normal lung, lung tumor and in the different lung cancer cell lines, suggesting nucleosomal nature of DNA (Figure 4). Primers designed to amplify products inside nucleosomes exhibited higher efficiency (banding intensity and/or qPCR values), than those primers that amplify bigger products and/or shifted to the left or right borders of nucleosomes. Furthermore, amplification efficiency differed among the lung cancer cell lines and was generally higher in cell lines which were less methylated and still expressed Cadm1. Such differences became more evident with the left or right border primers that in some cell lines amplification products were already absent. This result suggested differential nucleosome positioning among the different lung cancer cell lines. Overall, the highest amplification efficiency was observed in normal lung, then in lung tumor, and followed by the lung cancer cell lines.

Bottom Line: Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications.Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders.Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Environmental Hygiene, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany. reamon-buettner@item.fraunhofer.de

ABSTRACT
Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and 'bivalent' histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

Show MeSH
Related in: MedlinePlus